Oligomeric compounds comprising bicyclic nucleosides and uses thereof

ABSTRACT

The present invention provides oligomeric compounds. Certain such oligomeric compounds are useful for hybridizing to a complementary nucleic acid, including but not limited, to nucleic acids in a cell. In certain embodiments, hybridization results in modulation of the amount activity or expression of the target nucleic acid in a cell. In certain embodiments, the present invention provides compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise a region having a gapmer sugar motif. In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif.

BACKGROUND OF THE INVENTION

Antisense compounds have been used to modulate target nucleic acids.Antisense compounds comprising a variety of chemical modifications andmotifs have been reported. In certain instances, such compounds areuseful as research tools, diagnostic reagents, and as therapeuticagents. In certain instances antisense compounds have been shown tomodulate protein expression by binding to a target messenger RNA (mRNA)encoding the protein. In certain instances, such binding of an antisensecompound to its target mRNA results in cleavage of the mRNA. Antisensecompounds that modulate processing of a pre-mRNA have also beenreported. Such antisense compounds alter splicing, interfere withpolyadenlyation or prevent formation of the 5′-cap of a pre-mRNA.

Certain antisense compounds have been described previously. See forexample U.S. Pat. No. 7,399,845 and published International PatentApplication No. WO 2008/049085, which are hereby incorporated byreference herein in their entirety.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides compoundscomprising oligonucleotides. In certain embodiments, sucholigonucleotides comprise a region having a gapmer sugar motif. Incertain embodiments, such oligonucleotides consist of a region having agapmer sugar motif.

In certain embodiments, oligonucleotides have a sugar motif selectedfrom among:

BBB-(D)₈-BBBAA; KKK-(D)₈-KKKAA; LLL-(D)₈-LLLAA; BBB-(D)₈-BBBEE;KKK-(D)₈-KKKEE; LLL-(D)₈-LLLEE; BBB-(D)₇-BBBAA; KKK-(D)₇-KKKAA;LLL-(D)₇-LLLAA; BBB-(D)₇-BBBEE; KKK-(D)₇-KKKEE; LLL-(D)₇-LLLEE;BBB-(D)₈-BBBAAA; KKK-(D)₈-KKKAAA; LLL-(D)₈-LLLAAA; BBB-(D)₈-BBBEEE;KKK-(D)₈-KKKEEE; LLL-(D)₈-LLLEEE; BBB-(D)₇-BBBAAA; KKK-(D)₇-KKKAAA;LLL-(D)₇-LLLAAA; BBB-(D)₇-BBBEEE; KKK-(D)₇-KKKEEE; LLL-(D)₇-LLLEEE;BABA-(D)₈-ABAB; KAKA-(D)₈-AKAK; LALA-(D)₈-ALAL; BEBE-(D)₈-EBEB;KEKE-(D)₈-EKEK; LELE-(D)₈-ELEL; BABA-(D)₇-ABAB; KAKA-(D)₇-AKAK;LALA-(D)₇-ALAL; BEBE-(D)₇-EBEB; KEKE-(D)₇-EKEK; LELE-(D)₇-ELEL;ABAB-(D)₈-ABAB; AKAK-(D)₈-AKAK; ALAL-(D)₈-ALAL; EBEB-(D)₈-EBEB;EKEK-(D)₈-EKEK; ELEL-(D)₈-ELEL; ABAB-(D)₇-ABAB; AKAK-(D)₇-AKAK;ALAL-(D)₇-ALAL; EBEB-(D)₇-EBEB; EKEK-(D)₇-EKEK; ELEL-(D)₇-ELEL;AABB-(D)₈-BBAA; AAKK-(D)₈-KKAA; AALL-(D)₈-LLAA; EEBB-(D)₈-BBEE;EEKK-(D)₈-KKEE; EELL-(D)₈-LLEE; AABB-(D)₇-BBAA; AAKK-(D)₇-KKAA;AALL-(D)₇-LLAA; EEBB-(D)₇-BBEE; EEKK-(D)₇-KKEE; EELL-(D)₇-LLEE;BBB-(D)₉-BBA; KKK-(D)₉-KKA; LLL-(D)₉-LLA; BBB-(D)₉-BBE; KKK-(D)₉-KKE;LLL-(D)₉-LLE; BBB-(D)₈-BBA; KKK-(D)₈-KKA; LLL-(D)₈-LLA; BBB-(D)₈-BBE;KKK-(D)₈-KKE; LLL-(D)₈-LLE; BBB-(D)₇-BBA; KKK-(D)₇-KKA; LLL-(D)₇-LLA;BBB-(D)₇-BBE; KKK-(D)₇-KKE; LLL-(D)₇-LLE; ABBB-(D)₈-BBBA;AKKK-(D)₈-KKKA; ALLL-(D)₈-LLLA; EBBB-(D)₈-BBBE; EKKK-(D)₈-KKKE;ELLL-(D)₈-LLLE; ABBB-(D)₇-BBBA; AKKK-(D)₇-KKKA; ALLL-(D)₇-LLLA;EBBB-(D)₇-BBBE; EKKK-(D)₇-KKKE; ELLL-(D)₇-LLLE; ABBB-(D)₈-BBBAA;AKKK-(D)₈-KKKAA; ALLL-(D)₈-LLLAA; EBBB-(D)₈-BBBEE; EKKK-(D)₈-KKKEE;ELLL-(D)₈-LLLEE; ABBB-(D)₇-BBBAA; AKKK-(D)₇-KKKAA; ALLL-(D)₇-LLLAA;EBBB-(D)₇-BBBEE; EKKK-(D)₇-KKKEE; ELLL-(D)₇-LLLEE; AABBB-(D)₈-BBB;AAKKK-(D)₈-KKK; AALLL-(D)₈-LLL; EEBBB-(D)₈-BBB; EEKKK-(D)₈-KKK;EELLL-(D)₈-LLL; AABBB-(D)₇-BBB; AAKKK-(D)₇-KKK; AALLL-(D)₇-LLL;EEBBB-(D)₇-BBB; EEKKK-(D)₇-KKK; EELLL-(D)₇-LLL; AABBB-(D)₈-BBBA;AAKKK-(D)₈-KKKA; AALLL-(D)₈-LLLA; EEBBB-(D)₈-BBBE; EEKKK-(D)₈-KKKE;EELLL-(D)₈-LLLE; AABBB-(D)₇-BBBA; AAKKK-(D)₇-KKKA; AALLL-(D)₇-LLLA;EEBBB-(D)₇-BBBE; EEKKK-(D)₇-KKKE; EELLL-(D)₇-LLLE; ABBAABB-(D)₈-BB;AKKAAKK-(D)₈-KK; ALLAALLL-(D)₈-LL; EBBEEBB-(D)₈-BB; EKKEEKK-(D)₈-KK;ELLEELL-(D)₈-LL; ABBAABB-(D)₇-BB; AKKAAKK-(D)₇-KK; ALLAALL-(D)₇-LL;EBBEEBB-(D)₇-BB; EKKEEKK-(D)₇-KK; ELLEELL-(D)₇-LL; ABBABB-(D)₈-BBB;AKKAKK-(D)₈-KKK; ALLALLL-(D)₈-LLL; EBBEBB-(D)₈-BBB; EKKEKK-(D)₈-KKK;ELLELL-(D)₈-LLL; ABBABB-(D)₇-BBB; AKKAKK-(D)₇-KKK; ALLALL-(D)₇-LLL;EBBEBB-(D)₇-BBB; EKKEKK-(D)₇-KKK; ELLELL-(D)₇-LLL.

In certain embodiments, such oligonucleotides provide desirableproperties as therapeutic agents.

DETAILED DESCRIPTION OF THE INVENTION

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.Nucleosides are capable of being linked together to form an oligomericcompound, which is capable of hybridization to a complementaryoligomeric compound. In certain embodiments such complementaryoligomeric compound is a naturally occurring nucleic acid.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Inreference to an oligonucleotide, chemical modification does not includedifferences only in nucleobase sequence. Chemical modifications ofoligonucleotides include nucleoside modifications (including sugarmoiety modifications and nucleobase modifications) and internucleosidelinkage modifications.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moietyor a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is nota naturally occurring sugar moiety. Substituted sugar moieties include,but are not limited to furanosyls comprising substituents at the2′-position, the 3′-position, the 5′-position and/or the 4′-position.Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside is capable of (1) incorporation into an oligonucleotide and(2) hybridization to a complementary nucleoside. Such structures includerings comprising a different number of atoms than furanosyl (e.g., 4, 6,or 7-membered rings); replacement of the oxygen of a furanosyl with anon-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change inthe number of atoms and a replacement of the oxygen. Such structures mayalso comprise substitutions corresponding to those described forsubstituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugarsurrogates optionally comprising additional substituents). Sugarsurrogates also include more complex sugar replacements (e.g., thenon-ring systems of peptide nucleic acid). Sugar surrogates includewithout limitation morpholinos, cyclohexenyls and cyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified.

As used herein, “heterocyclic base” or “heterocyclic nucleobase” means anucleobase comprising a heterocyclic structure.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” means an atom or group of atoms bound to anoligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid.

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measurable activity” means ameasurable activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenlyation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound hybridizes.

As used herein, “single nucleotide polymorphism” or “SNP” means a singlenucleotide variation between the genomes of individuals of the samespecies. In some cases, a SNP may be a single nucleotide deletion orinsertion.

As used herein, “mRNA” means an RNA molecule that encodes a protein.

As used herein, “pre-mRNA” means an RNA transcript that has not beenfully processed into mRNA. Pre-RNA includes one or more intron.

As used herein, “object RNA” means an RNA molecule other than a targetRNA, the amount, activity, splicing, and/or function of which ismodulated, either directly or indirectly, by a target nucleic acid. Incertain embodiments, a target nucleic acid modulates splicing of anobject RNA. In certain such embodiments, an antisense compound modulatesthe amount or activity of the target nucleic acid, resulting in a changein the splicing of an object RNA and ultimately resulting in a change inthe activity or function of the object RNA.

As used herein, “microRNA” means a naturally occurring, small,non-coding RNA that represses gene expression of at least one mRNA. Incertain embodiments, a microRNA represses gene expression by binding toa target site within a 3′ untranslated region of an mRNA. In certainembodiments, a microRNA has a nucleobase sequence as set forth inmiRBase, a database of published microRNA sequences found athttp://microrna.sanger.ac.uk/sequences/. In certain embodiments, amicroRNA has a nucleobase sequence as set forth in miRBase version 12.0released September 2008, which is herein incorporated by reference inits entirety.

As used herein, “targeting” or “targeted to” means the association of anantisense compound to a particular target nucleic acid molecule or aparticular region of a target nucleic acid molecule. An antisensecompound targets a target nucleic acid if it is sufficientlycomplementary to the target nucleic acid to allow hybridization underphysiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity under stringent conditions. Complementary oligomericcompounds need not have nucleobase complementarity at each nucleoside.Rather, some mismatches are tolerated. In certain embodiments,complementary oligomeric compounds or regions are complementary at 70%of the nucleobases (70% complementary). In certain embodiments,complementary oligomeric compounds or regions are 80% complementary. Incertain embodiments, complementary oligomeric compounds or regions are90% complementary. In certain embodiments, complementary oligomericcompounds or regions are 95% complementary. In certain embodiments,complementary oligomeric compounds or regions are 100% complementary.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an antisense oligonucleotide specifically hybridizes tomore than one target site.

As used herein, “fully complementary” in reference to an oligonucleotideor portion thereof means that each nucleobase of the oligonucleotide orportion thereof is capable of pairing with a nucleobase of acomplementary nucleic acid or contiguous portion thereof. Thus, a fullycomplementary region comprises no mismatches or unhybridized nucleobasesin either strand.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “motif” means a pattern of chemical modifications in anoligomeric compound or a region thereof. Motifs may be defined bymodifications at certain nucleosides and/or at certain linking groups ofan oligomeric compound.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligomeric compound or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligomeric compound or region thereof. The nucleosides of such anoligomeric compound may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleoside have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “separate regions” means portions of an oligonucleotidewherein the chemical modifications or the motif of chemicalmodifications of any neighboring portions include at least onedifference to allow the separate regions to be distinguished from oneanother.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substituent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentinvention have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms differs from the atomor a group of atoms normally present in the named functional group. Incertain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino (═NR_(bb)), amido (—C(O)N(R_(bb))(R_(cc)) or—N(R_(bb))C(O)R_(a)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))—(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S—(O)₂R_(bb)).Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, anoptionally linked chemical functional group or a further substituentgroup with a preferred list including without limitation, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl. Selected substituentswithin the compounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butyryl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or poly-cyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

Oligomeric Compounds

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, such oligomeric compounds compriseoligonucleotides optionally comprising one or more conjugate and/orterminal groups. In certain embodiments, an oligomeric compound consistsof an oligonucleotide. In certain embodiments, oligonucleotides compriseone or more chemical modifications. Such chemical modifications includemodifications one or more nucleoside (including modifications to thesugar moiety and/or the nucleobase) and/or modifications to one or moreinternucleoside linkage.

Certain Sugar Moieties

In certain embodiments, oligomeric compounds of the invention compriseone or more modified nucleosides comprising a modified sugar moiety.Such oligomeric compounds comprising one or more sugar-modifiednucleosides may have desirable properties, such as enhanced nucleasestability or increased binding affinity with a target nucleic acidrelative to oligomeric compounds comprising only nucleosides comprisingnaturally occurring sugar moieties. In certain embodiments, modifiedsugar moieties are substituted sugar moieties. In certain embodiments,modified sugar moieties are sugar surrogates. Such sugar surrogates maycomprise one or more substitutions corresponding to those of substitutedsugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certainembodiments, substituted sugars comprise more than one non-bridgingsugar substituent, for example, 2′-F-5′-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—;4′-CH₂-2′,4′-(CH₂)₂-2′,4′-(CH₂)₃-2′. 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, andanalogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15,2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g.,WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, e.g., WO2008/150729, published Dec. 11, 2008);4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004);4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008) and4′-CH₂—O—CH₂-2′.

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B)13-D-Methyleneoxy (4′-CH₂—O-2′) BNA (also referred to as locked nucleicacid or LNA), (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F)Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to asconstrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H)methylene-amino (4′-CH2-N(R)-2′) BNA, (I) methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA,and (M) 4′-CH₂—O—CH₂-2′ as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfer, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surrogates comprise a 4′-sulfer atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran. Such tetrahydropyrans may befurther modified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′,2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

Certain Nucleobases

In certain embodiments, nucleosides of the present invention compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present invention comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH₃) uracil and cytosine and other alkynyl derivatives of pyrimidinebases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine,3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases,promiscuous bases, size-expanded bases, and fluorinated bases as definedherein. Further modified nucleobases include tricyclic pyrimidines suchas phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

Certain Internucleoside Linkages

In certain embodiments, the present invention provides oligomericcompounds comprising linked nucleosides. In such embodiments,nucleosides may be linked together using any internucleoside linkage.The two main classes of internucleoside linking groups are defined bythe presence or absence of a phosphorus atom. Representative phosphoruscontaining internucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of the oligomericcompound. In certain embodiments, internucleoside linkages having achiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Certain Motifs

In certain embodiments, the present invention provides oligomericcompounds comprising oligonucleotides. In certain embodiments, sucholigonucleotides comprise one or more chemical modification. In certainembodiments, chemically modified oligonucleotides comprise one or moremodified sugars. In certain embodiments, chemically modifiedoligonucleotides comprise one or more modified nucleobases. In certainembodiments, chemically modified oligonucleotides comprise one or moremodified internucleoside linkages. In certain embodiments, thechemically modifications (sugar modifications, nucleobase modifications,and/or linkage modifications) define a pattern or motif. In certainembodiments, the patterns of chemical modifications of sugar moieties,internucleoside linkages, and nucleobases are each independent of oneanother. Thus, an oligonucleotide may be described by its sugarmodification motif, internucleoside linkage motif and/or nucleobasemodification motif (as used herein, nucleobase modification motifdescribes the chemical modifications to the nucleobases independent ofthe sequence of nucleobases).

Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar modification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar modification motif, which comprises twoexternal regions or “wings” and an internal region or “gap.” The threeregions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar modification motifs of the two wings are the sameas one another (symmetric gapmer). In certain embodiments, the sugarmodification motifs of the 5′-wing differs from the sugar modificationmotif of the 3′-wing (asymmetric gapmer).

Certain 5′-Wings

In certain embodiments, the 5′-wing of a gapmer consists of 1 to 5linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 2 to 5 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 4 or 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of1 to 4 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 to 3 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 1 or 2 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 2 to 4 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of2 or 3 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 3 or 4 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 1 nucleoside. In certainembodiments, the 5′-wing of a gapmer consists of 2 linked nucleosides.In certain embodiments, the 5′-wing of a gapmer consists of 3 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 5 linked nucleosides.

In certain embodiments, the 5′-wing of a gapmer has a sugar motifselected from among those listed in the following non-limiting table:

TABLE 1 Certain 5′-Wing Sugar Motifs 5′-wing sugar motif # motif 1a BBB2a ABBB 3a AABB 4a BABA 5a ABAB 6a ABB 7a AABBB 8a ABBAABB 9a ABBABB 1bBBB 2b EBBB 3b EEBB 4b BEBE 5b EBEB 6b EBB 7b EEBBB 8b EBBEEBB 9b EBBEBB1c LLL 2c ALLL 3c AALL 4c LALA 5c ALAL 6c ALL 7c AALLL 8c ALLAALL 9cALLALL 1d LLL 2d ELLL 3d EELL 4d LELE 5d ELEL 6d ELL 7d EELLL 8d ELLEELL9d ELLELL 1e KKK 2e AKKK 3e AAKK 4e KAKA 5e AKAK 6e AKK 7e AAKKK 8eAKKAAKK 9e AKKAKK 1f KKK 2f EKKK 3f EEKK 4f KEKE 5f EKEK 6f EKK 7f EEKKK8f EKKEEKK 9f EKKEKK In the above table, “A” represents a modifiednucleoside comprising a non-bicyclic sugar moiety. In certainembodiments, such “A” nucleosides comprise a 2′-substituted sugarmoiety; “B” represents a bicyclic nucleoside; “K” represents aconstrained ethyl nucleoside; “L” represents an LNA nucleoside; and “E”represents a 2′-MOE nucleoside.

Certain 3′-Wings

In certain embodiments, the 3′-wing of a gapmer consists of 1 to 5linked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 2 to 5 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 4 or 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of1 to 4 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 to 3 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 1 or 2 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 2 to 4 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of2 or 3 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 3 or 4 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 1 nucleoside. In certainembodiments, the 3′-wing of a gapmer consists of 2 linked nucleosides.In certain embodiments, the 3′-wing of a gapmer consists of 3 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 5 linked nucleosides.

In certain embodiments, the 3′-wing of a gapmer has a sugar motifselected from among those listed in the following non-limiting table:

TABLE 2 Certain 3′-Wing Sugar Motifs 3′-wing sugar motif # motif 1a BBB2a BBBA 3a BBBAA 4a BBBAAA 5a BBAA 6a ABAB 7a BBA 8a BBA 1b BBB 2b BBBE3b BBBEE 4b BBBEEE 5b BBEE 6b EBEB 7b BBE 8b BBE 1c LLL 2c LLLA 3c LLLAA4c LLLAAA 5c LLAA 6c ALAL 7c LLA 8c LLA 1d LLL 2d LLLE 3d LLLEE 4dLLLEEE 5d LLEE 6d ELEL 7d LLE 8d LLE 1e KKK 2e KKKA 3e KKKAA 4e KKKAAA5e KKAA 6e AKAK 7e KKA 8e KKA 1f KKK 2f KKKE 3f KKKEE 4f KKKEEE 5f KKEE6f EKEK 7f KKE 8f KKE In the above table, “A” represents a modifiednucleoside comprising a modified non-bicyclic sugar moiety. In certainembodiments, such “A” nucleosides comprise a 2′-substituted sugarmoiety; “B” represents a bicyclic nucleoside; “K” represents aconstrained ethyl nucleoside; “L” represents an LNA nucleoside; and “E”represents a 2′-MOE nucleoside.

In certain embodiments, an oligonucleotide comprises any 3′-wing motifprovided herein. In certain such embodiments, the oligonucleotide is a3′-hemimer (does not comprise a 5′-wing). In certain embodiments, suchan oligonucleotide is a gapmer. In certain such embodiments, the 5′-wingof the gapmer may comprise any sugar modification motif.

Certain Gaps

In certain embodiments, the gap of a gapmer consists of 6 to 20 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6to 15 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 to 12 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 6 to 10 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 6 to 8 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6or 7 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 7 to 10 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 7 to 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 8 to 10 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 8or 9 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 linked nucleosides. In certain embodiments, the gap of agapmer consists of 7 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 8 linked nucleosides. In certain embodiments,the gap of a gapmer consists of 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 10 linked nucleosides. Incertain embodiments, the gap of a gapmer consists of 11 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 12linked nucleosides.

In certain embodiments, each nucleotide of the gap of a gapmer is a2′-deoxynucleoside. In certain embodiments, the gap comprises one ormore modified nucleosides. In certain embodiments, each nucleotide ofthe gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleosidethat is “DNA-like.” In such embodiments, “DNA-like” means that thenucleoside has similar characteristics to DNA, such that a duplexcomprising the gapmer and an RNA molecule is capable of activating RNaseH. For example, under certain conditions, 2′-fluoro (arabino)nucleosides (also referred to as FANA) have been shown to support RNaseH activation, and thus is DNA-like. In certain embodiments, one or morenucleosides of the gap of a gapmer is not a 2′-deoxynucleoside and isnot DNA-like. In certain such embodiments, the gapmer nonethelesssupports RNase H activation (e.g., by virtue of the number or placementof the non-DNA nucleosides).

Certain Gapmer Motifs

In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′wing, wherein the 5′-wing, gap, and 3′ wing are independently selectedfrom among those discussed above.

In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′wing, independently selected from among those proved in the abovetables, for example as provided in the following table:

TABLE 3 Certain Gapmer Sugar Motifs Gapmer 5-wing sugar motif 3′-wingsugar motif motif # (from table 1) Gap (from table 2) 1 1a, b, c, d, e,or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d, e, or f 2 2a, b, c, d,e, or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d, e, or f 3 3a, b, c,d, e, or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d, e, or f 4 4a, b,c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d, e, or f 5 5a,b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d, e, or f 66a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d, e, or f7 7a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d, e, orf 8 8a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d, e,or f 9 9a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 1a, b, c, d,e, or f 10 1a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 2a, b, c,d, e, or f 11 2a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 2a, b,c, d, e, or f 12 3a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 2a,b, c, d, e, or f 13 4a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides2a, b, c, d, e, or f 14 5a, b, c, d, e, or f 7, 8, or 92′-deoxynucleosides 2a, b, c, d, e, or f 15 6a, b, c, d, e, or f 7, 8,or 9 2′-deoxynucleosides 2a, b, c, d, e, or f 16 7a, b, c, d, e, or f 7,8, or 9 2′-deoxynucleosides 2a, b, c, d, e, or f 17 8a, b, c, d, e, or f7, 8, or 9 2′-deoxynucleosides 2a, b, c, d, e, or f 18 9a, b, c, d, e,or f 7, 8, or 9 2′-deoxynucleosides 2a, b, c, d, e, or f 19 1a, b, c, d,e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b, c, d, e, or f 20 2a, b, c,d, e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b, c, d, e, or f 21 3a, b,c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b, c, d, e, or f 22 4a,b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b, c, d, e, or f 235a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b, c, d, e, or f24 6a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b, c, d, e,or f 25 7a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b, c, d,e, or f 26 8a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b, c,d, e, or f 27 9a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 3a, b,c, d, e, or f 28 1a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 4a,b, c, d, e, or f 29 2a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides4a, b, c, d, e, or f 30 3a, b, c, d, e, or f 7, 8, or 92′-deoxynucleosides 4a, b, c, d, e, or f 31 4a, b, c, d, e, or f 7, 8,or 9 2′-deoxynucleosides 4a, b, c, d, e, or f 32 5a, b, c, d, e, or f 7,8, or 9 2′-deoxynucleosides 4a, b, c, d, e, or f 33 6a, b, c, d, e, or f7, 8, or 9 2′-deoxynucleosides 4a, b, c, d, e, or f 34 7a, b, c, d, e,or f 7, 8, or 9 2′-deoxynucleosides 4a, b, c, d, e, or f 35 8a, b, c, d,e, or f 7, 8, or 9 2′-deoxynucleosides 4a, b, c, d, e, or f 36 9a, b, c,d, e, or f 7, 8, or 9 2′-deoxynucleosides 4a, b, c, d, e, or f 37 1a, b,c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 5a, b, c, d, e, or f 38 2a,b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 5a, b, c, d, e, or f 393a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 5a, b, c, d, e, or f40 4a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 5a, b, c, d, e,or f 41 5a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 5a, b, c, d,e, or f 42 6a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 5a, b, c,d, e, or f 43 7a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 5a, b,c, d, e, or f 44 8a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 5a,b, c, d, e, or f 45 9a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides5a, b, c, d, e, or f 46 1a, b, c, d, e, or f 7, 8, or 92′-deoxynucleosides 6a, b, c, d, e, or f 47 2a, b, c, d, e, or f 7, 8,or 9 2′-deoxynucleosides 6a, b, c, d, e, or f 48 3a, b, c, d, e, or f 7,8, or 9 2′-deoxynucleosides 6a, b, c, d, e, or f 49 4a, b, c, d, e, or f7, 8, or 9 2′-deoxynucleosides 6a, b, c, d, e, or f 50 5a, b, c, d, e,or f 7, 8, or 9 2′-deoxynucleosides 6a, b, c, d, e, or f 51 6a, b, c, d,e, or f 7, 8, or 9 2′-deoxynucleosides 6a, b, c, d, e, or f 52 7a, b, c,d, e, or f 7, 8, or 9 2′-deoxynucleosides 6a, b, c, d, e, or f 53 8a, b,c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 6a, b, c, d, e, or f 54 9a,b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 6a, b, c, d, e, or f 551a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 7a, b, c, d, e, or f56 2a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 7a, b, c, d, e,or f 57 3a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 7a, b, c, d,e, or f 58 4a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 7a, b, c,d, e, or f 59 5a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 7a, b,c, d, e, or f 60 6a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 7a,b, c, d, e, or f 61 7a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides7a, b, c, d, e, or f 62 8a, b, c, d, e, or f 7, 8, or 92′-deoxynucleosides 7a, b, c, d, e, or f 63 9a, b, c, d, e, or f 7, 8,or 9 2′-deoxynucleosides 7a, b, c, d, e, or f 64 1a, b, c, d, e, or f 7,8, or 9 2′-deoxynucleosides 8a, b, c, d, e, or f 65 2a, b, c, d, e, or f7, 8, or 9 2′-deoxynucleosides 8a, b, c, d, e, or f 66 3a, b, c, d, e,or f 7, 8, or 9 2′-deoxynucleosides 8a, b, c, d, e, or f 67 4a, b, c, d,e, or f 7, 8, or 9 2′-deoxynucleosides 8a, b, c, d, e, or f 68 5a, b, c,d, e, or f 7, 8, or 9 2′-deoxynucleosides 8a, b, c, d, e, or f 69 6a, b,c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 8a, b, c, d, e, or f 70 7a,b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 8a, b, c, d, e, or f 718a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 8a, b, c, d, e, or f72 9a, b, c, d, e, or f 7, 8, or 9 2′-deoxynucleosides 8a, b, c, d, e,or f

In certain embodiments, a gapmer comprises a 5′-wing selected from amongthe 5′-wings provided herein and any 3′-wing.

In certain embodiments, the gapmer oligonucleotides provided herein areuseful in therapy. In certain such embodiments, a sugar motif isselected to provide antisense activity. In such embodiments, theantisense compound is potent. In certain embodiments, an antisensecompound is well tolerated when administered to an animal. In certainembodiments, an antisense compound is active and potent and welltolerated. Therapeutic index is a measure of potency divided by ameasure of toxicity. In certain embodiments, therapeutic indicationinforms the relative importance of potency, activity, and tolerability.For example, for treatment of aggressive and lethal diseases, one mayselect a highly active and potent compound, even if its tolerability isnot ideal. For other indications, safety/tolerability may be paramount,even if it means selecting a compound that is not the mostactive/potent. Accordingly, in certain embodiments, antisense compoundscomprising the sugar motifs provided herein have therapeutic profilessuitable for a variety of indications.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₈-BBBAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₈-KKKAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₈-LLLAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₈-BBBEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₈-KKKEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₈-LLLEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₇-BBBAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₇-KKKAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₇-LLLAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₇-BBBEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₇-KKKEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₇-LLLEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₈-BBBAAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₈-KKKAAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₈-LLLAAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₈-BBBEEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₈-KKKEEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₈-LLLEEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₇-BBBAAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₇-KKKAAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₇-LLLAAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₇-BBBEEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₇-KKKEEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₇-LLLEEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BABA-(D)₈-ABAB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KAKA-(D)₈-AKAK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LALA-(D)₈-ALAL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BEBE-(D)₈-EBEB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KEKE-(D)₈-EKEK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LELE-(D)₈-ELEL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BABA-(D)₇-ABAB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KAKA-(D)₇-AKAK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LALA-(D)₇-ALAL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BEBE-(D)₇-EBEB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KEKE-(D)₇-EKEK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LELE-(D)₇-ELEL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABAB-(D)₈-ABAB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKAK-(D)₈-AKAK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALAL-(D)₈-ALAL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBEB-(D)₈-EBEB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKEK-(D)₈-EKEK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELEL-(D)₈-ELEL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABAB-(D)₇-ABAB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKAK-(D)₇-AKAK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALAL-(D)₇-ALAL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBEB-(D)₇-EBEB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKEK-(D)₇-EKEK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELEL-(D)₇-ELEL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AABB-(D)₈-BBAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AAKK-(D)₈-KKAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AALL-(D)₈-LLAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEBB-(D)₈-BBEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEKK-(D)₈-KKEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EELL-(D)₈-LLEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AABB-(D)₇-BBAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AAKK-(D)₇-KKAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AALL-(D)₇-LLAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEBB-(D)₇-BBEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEKK-(D)₇-KKEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EELL-(D)₇-LLEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₉-BBA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₉-KKA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₉-LLA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₉-BBE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₉-KKE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₉-LLE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₈-BBA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₈-KKA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₈-LLA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₈-BBE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₈-KKE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₈-LLE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₇-BBA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₇-KKA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₇-LLA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif BBB-(D)₇-BBE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif KKK-(D)₇-KKE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif LLL-(D)₇-LLE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABBB-(D)₈-BBBA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKKK-(D)₈-KKKA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALLL-(D)₈-LLLA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBBB-(D)₈-BBBE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKKK-(D)₈-KKKE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELLL-(D)₈-LLLE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABBB-(D)₇-BBBA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKKK-(D)₇-KKKA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALLL-(D)₇-LLLA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBBB-(D)₇-BBBE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKKK-(D)₇-KKKE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELLL-(D)₇-LLLE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABBB-(D)₈-BBBAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKKK-(D)₈-KKKAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALLL-(D)₈-LLLAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBBB-(D)₈-BBBEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKKK-(D)₈-KKKEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELLL-(D)₈-LLLEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABBB-(D)₇-BBBAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKKK-(D)₇-KKKAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALLL-(D)₇-LLLAA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBBB-(D)₇-BBBEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKKK-(D)₇-KKKEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELLL-(D)₇-LLLEE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AABBB-(D)₈-BBB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AAKKK-(D)₈-KKK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AALLL-(D)₈-LLL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEBBB-(D)₈-BBB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEKKK-(D)₈-KKK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EELLL-(D)₈-LLL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AABBB-(D)₇-BBB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AAKKK-(D)₇-KKK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AALLL-(D)₇-LLL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEBBB-(D)₇-BBB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEKKK-(D)₇-KKK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EELLL-(D)₇-LLL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AABBB-(D)₈-BBBA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AAKKK-(D)₈-KKKA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AALLL-(D)₈-LLLA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEBBB-(D)₈-BBBE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEKKK-(D)₈-KKKE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EELLL-(D)₈-LLLE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AABBB-(D)₇-BBBA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AAKKK-(D)₇-KKKA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AALLL-(D)₇-LLLA has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEBBB-(D)₇-BBBE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EEKKK-(D)₇-KKKE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EELLL-(D)₇-LLLE has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABBAABB-(D)₈-BB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKKAAKK-(D)₈-KK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALLAALLL-(D)₈-LL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBBEEBB-(D)₈-BB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKKEEKK-(D)₈-KK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELLEELL-(D)₈-LL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABBAABB-(D)₇-BB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKKAAKK-(D)₇-KK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALLAALL-(D)₇-LL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBBEEBB-(D)₇-BB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKKEEKK-(D)₇-KK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELLEELL-(D)₇-LL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABBABB-(D)₈-BBB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKKAKK-(D)₈-KKK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALLALLL-(D)₈-LLL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBBEBB-(D)₈-BBB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKKEKK-(D)₈-KKK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELLELL-(D)₈-LLL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ABBABB-(D)₇-BBB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif AKKAKK-(D)₇-KKK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ALLALL-(D)₇-LLL has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EBBEBB-(D)₇-BBB has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif EKKEKK-(D)₇-KKK has a desirable therapeutic profile.

In certain embodiments, an antisense oligonucleotide having a sugarmotif ELLELL-(D)₇-LLL has a desirable therapeutic profile.

In the above embodiments, A represents a modified nucleoside comprisinga non-bicyclic modified sugar. In certain embodiments, A comprises asubstituted sugar moiety. In certain embodiments, A comprises a2′-substituted sugar moiety.

In the above embodiments, B represents a modified nucleoside comprisinga bicyclic sugar moiety. In certain embodiments, B comprises a sugarmoiety comprising a 4′-2′ bridge. In certain such embodiments, the sugarmoiety of B is selected from: α-L-Methyleneoxy (4′-CH₂—O-2′),β-D-Methyleneoxy (4′-CH₂—O-2′), Ethyleneoxy (4′-(CH₂)₂—O-2′), Aminooxy(4′-CH₂—O—N(R)-2′), Oxyamino (4′-CH₂—N(R)—O-2′), Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′), methylene-thio (4′-CH₂—S-2′), methylene-amino(4′-CH2-N(R)-2′), methyl carbocyclic (4′-CH₂—CH(CH₃)-2′), propylenecarbocyclic (4′-(CH₂)₃-2′), and 4′-CH₂—O—CH₂-2′.

In the above embodiments, E represents a nucleoside having a 2′-MOE.

In the above embodiments, L represents an LNA nucleoside (i.e., abicyclic sugar comprising a 4′-CH₂—O-2′ bridge.

In the above embodiments, K represents an cEt nucleoside (i.e., abicyclic sugar comprising a 4′-CH(CH₃)—O-2′ bridge.

In the above embodiments, D represents a nucleoside having an unmodifiedDNA sugar moiety.

Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif, as described above for sugar modification motif. In suchembodiments, the internucleoside linkages in each of two wing regionsare different from the internucleoside linkages in the gap region. Incertain embodiments the internucleoside linkages in the wings arephosphodiester and the internucleoside linkages in the gap arephosphorothioate. The sugar modification motif is independentlyselected, so such oligonucleotides having a gapped internucleosidelinkage motif may or may not have a gapped sugar modification motif andif it does have a gapped sugar motif, the wing and gap lengths may ormay not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present invention comprise a region of uniformlymodified internucleoside linkages. In certain such embodiments, theoligonucleotide comprises a region that is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide is uniformly linked by phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate and at least oneinternucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certain suchembodiments, nucleobase modifications are arranged in a gapped motif. Incertain embodiments, nucleobase modifications are arranged in analternating motif. In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

Certain Overall Lengths

In certain embodiments, the present invention provides oligomericcompounds including oligonucleotides of any of a variety of ranges oflengths. In certain embodiments, the invention provides oligomericcompounds or oligonucleotides consisting of X to Y linked nucleosides,where X represents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≦Y. For example, in certainembodiments, the invention provides oligomeric compounds which compriseoligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linkednucleosides. In embodiments where the number of nucleosides of anoligomeric compound or oligonucleotide is limited, whether to a range orto a specific number, the oligomeric compound or oligonucleotide may,nonetheless further comprise additional other substituents. For example,an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotideshaving 31 nucleosides, but, unless otherwise indicated, such anoligonucleotide may further comprise, for example one or moreconjugates, terminal groups, or other substituents. In certainembodiments, a gapmer oligonucleotide has any of the above lengths.

In certain embodiments, any of the gapmer motifs provided above,including but not limited to gapmer motifs 1-278 provided in Tables 3and 4, may have any of the above lengths. One of skill in the art willappreciate that certain lengths may not be possible for certain motifs.For example: a gapmer having a 5′-wing region consisting of fournucleotides, a gap consisting of at least six nucleotides, and a 3′-wingregion consisting of three nucleotides cannot have an overall lengthless than 13 nucleotides. Thus, one would understand that the lowerlength limit is 13 and that the limit of 10 in “10-20” has no effect inthat embodiment.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range. For example, an oligonucleotide consisting of20-25 linked nucleosides comprising a 5′-wing consisting of 5 linkednucleosides; a 3′-wing consisting of 5 linked nucleosides and a centralgap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap(before reaching the overall length limitation of 25). Such additionalnucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing.

Certain Oligonucleotides

In certain embodiments, oligonucleotides of the present invention arecharacterized by their sugar motif, internucleoside linkage motif,nucleobase modification motif and overall length. In certainembodiments, such parameters are each independent of one another. Thus,each internucleoside linkage of an oligonucleotide having a gapmer sugarmotif may be modified or unmodified and may or may not follow the gapmermodification pattern of the sugar modifications. Thus, theinternucleoside linkages within the wing regions of a sugar-gapmer maybe the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region. Likewise,such sugar-gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.

Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamics, pharmacokinetics, stability, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligomericcompound, such as an oligonucleotide. Conjugate groups includes withoutlimitation, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groupshave been described previously, for example: cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

In certain embodiments, conjugate groups are directly attached tooligonucleotides in oligomeric compounds. In certain embodiments,conjugate groups are attached to oligonucleotides by a conjugate linkinggroup. In certain such embodiments, conjugate linking groups, including,but not limited to, bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Conjugate linkinggroups are useful for attachment of conjugate groups, such as chemicalstabilizing groups, functional groups, reporter groups and other groupsto selective sites in a parent compound such as for example anoligomeric compound. In general a bifunctional linking moiety comprisesa hydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such aschemical functional group or a conjugate group. In some embodiments, theconjugate linker comprises a chain structure or an oligomer of repeatingunits such as ethylene glycol or amino acid units. Examples offunctional groups that are routinely used in a bifunctional linkingmoiety include, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties includepyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′ end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group. In certain embodiments, thepresent invention provides oligomeric compounds. In certain embodiments,oligomeric compounds comprise an oligonucleotide. In certainembodiments, an oligomeric compound comprises an oligonucleotide and oneor more conjugate and/or terminal groups. Such conjugate and/or terminalgroups may be added to oligonucleotides having any of the chemicalmotifs discussed above. Thus, for example, an oligomeric compoundcomprising an oligonucleotide having region of alternating nucleosidesmay comprise a terminal group.

Antisense Compounds

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. Such antisense compounds are capable ofhybridizing to a target nucleic acid, resulting in at least oneantisense activity. In certain embodiments, antisense compoundsspecifically hybridize to one or more target nucleic acid. In certainembodiments, a specifically hybridizing antisense compound has anucleobase sequence comprising a region having sufficientcomplementarity to a target nucleic acid to allow hybridization andresult in antisense activity and insufficient complementarity to anynon-target so as to avoid non-specific hybridization to any non-targetnucleic acid sequences under conditions in which specific hybridizationis desired (e.g., under physiological conditions for in vivo ortherapeutic uses, and under conditions in which assays are performed inthe case of in vitro assays).

In certain embodiments, the present invention provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

Certain Antisense Activities and Mechanisms

In certain antisense activities, hybridization of an antisense compoundresults in recruitment of a protein that cleaves of the target nucleicacid. For example, certain antisense compounds result in RNase Hmediated cleavage of target nucleic acid. RNase H is a cellularendonuclease that cleaves the RNA strand of an RNA:DNA duplex. The “DNA”in such an RNA:DNA duplex, need not be unmodified DNA. In certainembodiments, the invention provides antisense compounds that aresufficiently “DNA-like” to elicit RNase H activity. Such DNA-likeantisense compounds include, but are not limited to gapmers havingunmodified deoxyfuronose sugar moieties in the nucleosides of the gapand modified sugar moieties in the nucleosides of the wings.

Antisense activities may be observed directly or indirectly. In certainembodiments, observation or detection of an antisense activity involvesobservation or detection of a change in an amount of a target nucleicacid or protein encoded by such target nucleic acid; a change in theratio of splice variants of a nucleic acid or protein; and/or aphenotypic change in a cell or animal.

In certain embodiments, compounds comprising oligonucleotides having agapmer motif described herein have desirable properties compared tonon-gapmer oligonucleotides or to gapmers having other motifs. Incertain circumstances, it is desirable to identify motifs resulting in afavorable combination of potent antisense activity and relatively lowtoxicity. In certain embodiments, compounds of the present inventionhave a favorable therapeutic index (measure of potency divided bymeasure of toxicity).

Certain Target Nucleic Acids

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid. In certain embodiments, the target nucleic acid is anendogenous RNA molecule. In certain embodiments, the target nucleic acidis a non-coding RNA. In certain such embodiments, the target non-codingRNA is selected from: a long-non-coding RNA, a short non-coding RNA, anintronic RNA molecule, a snoRNA, a scaRNA, a microRNA (includingpre-microRNA and mature microRNA), a ribosomal RNA, and promoterdirected RNA. In certain embodiments, the target nucleic acid encodes aprotein. In certain such embodiments, the target nucleic acid isselected from: an mRNA and a pre-mRNA, including intronic, exonic anduntranslated regions. In certain embodiments, oligomeric compounds areat least partially complementary to more than one target nucleic acid.For example, antisense compounds of the present invention may mimicmicroRNAs, which typically bind to multiple targets.

In certain embodiments, the target nucleic acid is a nucleic acid otherthan a mature mRNA. In certain embodiments, the target nucleic acid is anucleic acid other than a mature mRNA or a microRNA. In certainembodiments, the target nucleic acid is a non-coding RNA other than amicroRNA. In certain embodiments, the target nucleic acid is anon-coding RNA other than a microRNA or an intronic region of apre-mRNA. In certain embodiments, the target nucleic acid is a longnon-coding RNA. In certain embodiments, the target RNA is an mRNA. Incertain embodiments, the target nucleic acid is a pre-mRNA. In certainsuch embodiments, the target region is entirely within an intron. Incertain embodiments, the target region spans an intron/exon junction. Incertain embodiments, the target region is at least 50% within an intron.In certain embodiments, the target nucleic acid is selected from amongnon-coding RNA, including exonic regions of pre-mRNA. In certainembodiments, the target nucleic acid is a ribosomal RNA (rRNA). Incertain embodiments, the target nucleic acid is a non-coding RNAassociated with splicing of other pre-mRNAs. In certain embodiments, thetarget nucleic acid is a nuclear-retained non-coding RNA.

In certain embodiments, antisense compounds described herein arecomplementary to a target nucleic acid comprising a single-nucleotidepolymorphism. In certain such embodiments, the antisense compound iscapable of modulating expression of one allele of the single-nucleotidepolymorphism-containing-target nucleic acid to a greater or lesserextent than it modulates another allele. In certain embodiments anantisense compound hybridizes to a single-nucleotidepolymorphism-containing-target nucleic acid at the single-nucleotidepolymorphism site. In certain embodiments an antisense compoundhybridizes to a single-nucleotide polymorphism-containing-target nucleicacid near the single-nucleotide polymorphism site. In certainembodiments, the target nucleic acid is a Huntingtin gene transcript. Incertain embodiments, the target nucleic acid is a single-nucleotidepolymorphism-containing-target nucleic acid other than a Huntingtin genetranscript. In certain embodiments, the target nucleic acid is anynucleic acid other than a Huntingtin gene transcript.

Certain Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active antisense oligomericcompound.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present invention provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human.

In certain embodiments, the present invention provides methods ofadministering a pharmaceutical composition comprising an oligomericcompound of the present invention to an animal. Suitable administrationroutes include, but are not limited to, oral, rectal, transmucosal,intestinal, enteral, topical, suppository, through inhalation,intrathecal, intracerebroventricular, intraperitoneal, intranasal,intraocular, intratumoral, and parenteral (e.g., intravenous,intramuscular, intramedullary, and subcutaneous). In certainembodiments, pharmaceutical intrathecals are administered to achievelocal rather than systemic exposures.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

EXAMPLES

The following examples illustrate certain embodiments of the presentinvention and are not limiting. Moreover, where specific embodiments areprovided, the inventors have contemplated generic application of thosespecific embodiments. For example, disclosure of an oligonucleotidehaving a particular motif provides reasonable support for additionaloligonucleotides having the same or similar motif. And, for example,where a particular high-affinity modification appears at a particularposition, other high-affinity modifications at the same position areconsidered suitable, unless otherwise indicated.

Where nucleobase sequences are not provided, to allow assessment of therelative effects of nucleobase sequence and chemical modification,throughout the examples, oligomeric compounds are assigned a “SequenceCode.” Oligomeric compounds having the same Sequence Code have the samenucleobase sequence. Oligomeric compounds having different SequenceCodes have different nucleobase sequences.

Example 1: Antisense Inhibition of Human Target X in HuVEC Cells

Antisense oligonucleotides were designed to target a messenger RNAmolecule (Target X) and were tested for their effects on target X mRNAin vitro. Cultured HuVEC cells at a density of 20,000 cells per wellwere transfected using electroporation with 250 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and Target X mRNA levels were measured byquantitative real-time PCR. Target X mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN®. Results are presentedas percent inhibition of Target X, relative to untreated control cells.A total of 40 oligonucleotides were tested. Only those oligonucleotideswhich were selected for further study are shown in Table 4.

The ‘Chemistry’ column describes the sugar modifications of eacholigonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the numberindicates the number of deoxynucleosides; and ‘e’ indicates a MOEmodification. The internucleoside linkages throughout each gapmer arephosphorothioate linkages. All cytosine residues throughout each gapmerare 5-methylcytosines. “Start site” indicates the 5′-most nucleoside towhich the gapmer is targeted in the human gene sequence of Target X.“Stop site” indicates the 3′-most nucleoside to which the gapmer istargeted human gene sequence of Target X. Each gapmer listed in Table 4is targeted to the human Target X genomic sequence. Oligonucleotideshaving the same start site and stop site as one another have theidentical nucleobase sequence.

TABLE 4 Target Target % Start Site Stop Site ISIS No Chemistryinhibition 58721 58736 549457 kkk-10-kkk 67 58751 58766 58722 58737549458 kkk-10-kkk 71 58752 58767 58720 58735 560098 kkk-10-kkk 69 5875058765 58751 58766 58721 58736 560131 kkk-9-kkke 74 58751 58766 5872158736 560137 ekkk-8-kkke 66 58751 58766 58750 58765 58720 58735 569213kkk-9-kkke 69 58750 58765 58720 58735 569216 ekkk-8-kkke 68 58750 5876558721 58736 569222 eekkk-8-kkk 74 58751 58766 58721 58736 569228eekkk-7-kkke 67 58751 58766 58720 58735 569236 ekkk-7-kkkee 66 5875058765

Example 2: Dose-Dependent Antisense Inhibition of Human Target X inHuVEC Cells

Gapmers from the studies described above exhibiting significant in vitroinhibition of Target X mRNA were selected and tested at various doses inHuVEC cells. Cells were plated at a density of 20,000 cells per well andtransfected using electroporation with 31.3 μM, 62.5 μM, 125.0 μM, 250.0μM, 500.0 μM, and 1000.0 μM concentrations of antisense oligonucleotide,as specified in Table 5. After a treatment period of approximately 16hours, RNA was isolated from the cells and Target X mRNA levels weremeasured by quantitative real-time PCR. Target X mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of Target X, relative tountreated control cells. The antisense oligonucleotides were tested in aseries of experiments that had similar culture conditions. The resultsfor each experiment are presented in separate tables shown below.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 5. As illustrated, Target X mRNA levels werereduced in a dose-dependent manner in the antisense oligonucleotidetreated cells.

TABLE 5 31.25 62.5 125.0 250.0 500.0 1000.0 IC₅₀ ISIS No nM nM nM nM nMnM (μM) 549457 34 44 75 82 93 96 0.06 549458 30 36 54 70 85 90 0.10560098 30 54 65 78 89 97 0.07 560131 16 48 65 82 89 97 0.09 560137 35 3964 73 89 94 0.08 569213 35 53 65 83 94 96 0.06 569216 38 51 68 83 91 960.05 569222 36 48 67 83 91 98 0.06 569228 26 43 62 78 88 92 0.09 56923617 39 54 79 84 92 0.11

Example 3: Dose-Dependent Antisense Inhibition of Human Target X inHuVEC Cells

Additional antisense oligonucleotides were designed as deoxy, MOE and(S)-cEt oligonucleotides targeting Target X gene sequences and weretested at various doses in HuVEC cells. The ‘Chemistry’ column describesthe sugar modifications of each oligonucleotide. ‘k’ indicates an(S)-cEt sugar modification; the number indicates the number ofdeoxynucleosides; otherwise indicates deoxyribose; and ‘e’ indicates aMOE modification. The internucleoside linkages throughout each gapmerare phosphorothioate linkages. All cytosine residues throughout eachgapmer are 5-methylcytosines. “Start site” indicates the 5′-mostnucleoside to which the gapmer is targeted in the human gene sequence.“Stop site” indicates the 3′-most nucleoside to which the gapmer istargeted human gene sequence. Each gapmer listed in Table 6 is targetedto the human Target X genomic sequence. Oligonucleotides having the samestart site and stop site as one another have the identical nucleobasesequence.

TABLE 6 Target Target Start Site Stop Site ISIS No Chemistry 58720 58735569221 eekkk-8-kkk 58750 58765 58720 58735 569227 eekkk-7-kkke 5875058765 58720 58735 569236 ekkk-7-kkkee 58750 58765 58720 58735 579666ekkeekk-7-kk 58750 58765 58721 58736 579667 ekkeekk-7-kk 58751 5876658720 58735 579670 ekkekk-7-kkk 58750 58765 58721 58736 579671ekkekk-7-kkk 58751 58766 58721 58736 569228 eekkk-7-kkke 58751 5876658723 58738 579669 ekkeekk-7-kk 58753 58768 58722 58737 579672ekkekk-7-kkk 58752 58767 58722 58737 569217 ekkk-8-kkke 58752 5876758723 58738 569214 kkk-9-kkke 58753 58768 58723 58738 560099 kkk-10-kkk58753 58768

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 62.5 μM, 125.0 μM, 250.0 μM, 500.0 μM, and1000.0 μM concentrations of antisense oligonucleotide, as specified inTables 10-12. After a treatment period of approximately 16 hours, RNAwas isolated from the cells and Target X mRNA levels were measured byquantitative real-time PCR. Target X mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN®. Results are presentedas percent inhibition of Target X, relative to untreated control cells.The antisense oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Tables 7-9. As illustrated, Target X mRNA levelswere reduced in a dose-dependent manner in some of the antisenseoligonucleotide treated cells.

TABLE 7 62.5 125.0 250.0 500.0 1000.0 IC₅₀ ISIS No nM nM nM nM nM (nM)549458 25 46 55 64 78 203 569227 8 40 33 51 73 388 569228 29 44 63 77 87158 569236 4 35 54 68 88 252 579666 33 34 47 64 80 229 579667 30 29 4436 76 411

TABLE 8 62.5 125.0 250.0 500.0 1000.0 IC₅₀ ISIS No nM nM nM nM nM (nM)549458 16 22 44 64 74 324 579669 24 39 45 74 91 207 579670 27 28 55 7570 236 579671 6 40 54 57 77 288 579672 9 30 50 72 86 258

TABLE 9 62.5 125.0 250.0 500.0 1000.0 IC₅₀ ISIS No nM nM nM nM nM (nM)549458 19 22 45 38 71 470 569214 20 26 61 62 76 265 569217 34 39 49 6464 247 569221 12 32 59 57 73 294

Example 4: Antisense Inhibition of Human Target X in HuVEC Cells

Additional antisense oligonucleotides were designed targeting Target Xnucleic acid and were tested for their effects on Target X mRNA invitro. Cultured HuVEC cells at a density of 20,000 cells per well weretransfected using electroporation with 1,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and Target X mRNA levels were measured byquantitative real-time PCR. Target X mRNA levels were adjusted accordingto total RNA content, as measured by RIBOGREEN®. Results are presentedas percent inhibition of Target X, relative to untreated control cells.A total of 75 oligonucleotides were tested. Only those oligonucleotideswhich were selected for further study are shown in Table 10.

The ‘Chemistry’ column describes the sugar modifications of eacholigonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the numberindicates the number of deoxynucleosides; otherwise ‘d.’ indicatesdeoxyribose; and ‘e’ indicates a MOE modification. The internucleosidelinkages throughout each gapmer are phosphorothioate linkages. Allcytosine residues throughout each gapmer are 5-methylcytosines.

The “Start site” indicates the 5′-most nucleoside to which the gapmer istargeted in the human gene sequence. “Stop site” indicates the 3′-mostnucleoside to which the gapmer is targeted human gene sequence. Eachgapmer listed in Table 10 is targeted to the human Target X genomicsequence. Oligonucleotides having the same start site and stop site asone another have the identical nucleobase sequence.

TABLE 10 Target Target % Start Site Stop Site ISIS No Chemistryinhibition 5062 5077 549372 kkk-10-kkk 64 5061 5076 585233 kkk-8-keeee69 5062 5077 585259 ekkk-9-kkk 71 5062 5077 585262 kkk-9-kkke 77 50625077 585263 kkk-8-kkkee 69 5062 5077 585264 kkk-7-kkkeee 62 5062 5077585265 eekk-8-kkee 69 5062 5077 585268 keke-8-ekek 72 5062 5077 585269ekek-8-ekek 73 5062 5077 585271 ekk-10-kke 57 5062 5077 585274kkk-10-kke 65 58719 58734 586124 kkk-10-kkk 82 58720 58735 569227eekkk-7-kkke 51 58750 58765 58722 58737 560132 kkk-9-kkke 58 58752 5876758722 58737 569229 eekkk-7-kkke 57 58752 58767 58722 58737 569238ekkk-7-kkkee 51 58752 58767 58722 58737 549458 kkk-10-kkk 87 58752 5876758722 58737 569223 eekkk-8-kkk 59 58752 58767 58724 58739 569215kkk-9-kkke 59 58754 58769 58725 58740 560133 kkk-9-kkke 53 58755 5877058725 58740 569220 ekkk-8-kkke 58 58755 58770 58721 58736 586224kkkkk-8-kkk 90 58751 58766 58722 58737 586225 kkkkk-8-kkk 88 58752 5876758720 58735 586227 kkkkk-8-kkk 87 58750 58765

Example 5: Dose-Dependent Antisense Inhibition of Human Target X inHuVEC Cells

Antisense oligonucleotides from the studies described above exhibitingsignificant in vitro inhibition of Target X mRNA were selected andtested at various doses in HuVEC cells. Cells were plated at a densityof 20,000 cells per well and transfected using electroporation with31.25 μM, 62.5 μM, 125.0 μM, 250.0 μM, 500.0 μM, and 1000.0 μMconcentrations of antisense oligonucleotide, as specified in Table 11.After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and Target X mRNA levels were measured by quantitativereal-time PCR. Target X mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of Target X, relative to untreated control cells. Theantisense oligonucleotides were tested in a series of experiments thathad similar culture conditions. The results for each experiment arepresented in separate tables shown below.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 11. As illustrated, Target X mRNA levels werereduced in a dose-dependent manner in the antisense oligonucleotidetreated cells.

TABLE 11 31.25 62.5 125.0 250.0 500.0 1000.0 IC₅₀ ISIS No nM nM nM nM nMnM nM 549372 2 17 31 51 61 80 271 549458 0 19 40 63 74 90 196 560132 819 21 53 65 85 252 560133 17 15 24 35 58 79 336 569215 12 2 26 55 71 90234 569220 11 29 34 43 59 78 275 569223 21 20 30 59 73 87 191 569227 1322 45 46 61 74 255 569229 16 14 36 47 74 84 220 569238 4 32 33 54 71 88202

Example 6: Dose-Dependent Antisense Inhibition of Human Target X inHuVEC Cells

Gapmers from Example 8 exhibiting significant in vitro inhibition ofTarget X mRNA were selected and tested at various doses in HuVEC cells.Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 46.9 μM, 187.5 μM, 750.0 μM, and 3000.0 μMconcentrations of antisense oligonucleotide, as specified in Table 12.After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and Target X mRNA levels were measured by quantitativereal-time PCR. Target X mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of AR, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 12. As illustrated, Target X mRNA levels werereduced in a dose-dependent manner in antisense oligonucleotide treatedcells.

TABLE 12 46.9 187.5 750.0 3000.0 IC₅₀ ISIS No nM nM nM nM (μM) 549372 941 66 87 0.29 549458 15 50 85 96 0.19 586124 28 47 84 94 0.13 586224 3975 93 98 0.05 586225 17 61 89 97 0.13 586227 20 60 88 96 0.13

Example 7: Tolerability of Antisense Oligonucleotides Targeting HumanTarget X in CD1 Mice

CD1® mice (Charles River, Mass.) are a multipurpose mice model,frequently utilized for safety and efficacy testing. The mice weretreated with ISIS antisense oligonucleotides presented in Table 13 andevaluated for changes in the levels of various plasma chemistry markers.

TABLE 13 ISIS No Chemistry 549372 kkk-10-kkk 585233 kkk-8-keeee 585259ekkk-9-kkk 585262 kkk-9-kkke 585263 kkk-8-kkkee 585264 kkk-7-kkkeee585265 eekk-8-kkee 585268 keke-8-ekek 585269 ekek-8-ekekTreatment

Groups of 4-6-week old male CD1 mice were injected subcutaneously twicea week for 4 weeks with 100 mg/kg of ISIS 549372, ISIS 585233, ISIS585259, ISIS 585262, ISIS 585263, ISIS 585264, ISSI 585265, ISSI 585268,ISIS 585269, ISIS 585271, or ISIS 585274 (weekly dose of 200 mg/kg). Onegroup of 4-6-week old male CD1 mice was injected subcutaneously twice aweek for 4 weeks with PBS. Mice were euthanized 48 hours after the lastdose, and organs and plasma were harvested for further analysis.

Organ Weights

To evaluate the effect of ISIS oligonucleotides on organ weights, micewere euthanized and the liver, kidney and spleen for mice from allgroups were weighed. The results are presented in Table 14. ISISoligonucleotides that caused changes in the weights of any of the organsoutside the expected range for antisense oligonucleotides were excludedin further studies.

TABLE 14 Organ weights Liver Kidney Spleen PBS 201 64 15 ISIS 549372 24452 45 ISIS 585233 241 46 14 ISIS 585259 261 49 15 ISIS 585262 257 57 35ISIS 585263 206 52 14 ISIS 585264 237 50 17 ISIS 585265 219 45 14 ISIS585268 218 50 21 ISIS 585269 214 48 16Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, albumin,cholesterol, and BUN were measured using an automated clinical chemistryanalyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results arepresented in Table 15. ISIS oligonucleotides that caused changes in thelevels of any of the liver or kidney function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 15 Plasma chemistry markers in CD1 mice plasma Albu- Choles- ALTAST min BUN terol Bilirubin (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL)PBS 40 62 2.6 26 112 0.19 ISIS 549372 116 151 2 25.4 40 0.16 ISIS 585233884 716 1.8 23 93 0.94 ISIS 585259 2858 1302 2.3 24.4 93 0.39 ISIS585262 860 823 2 25.1 48 0.25 ISIS 585263 133 113 2.4 28.3 82 0.21 ISIS585264 134 108 2.2 28.2 109 0.17 ISIS 585265 404 229 2 24.5 98 0.19 ISIS585268 105 140 2.1 23.7 75 0.13 ISIS 585269 354 202 2.1 25.8 94 0.15Hematology Assays

Blood obtained from all mice groups were analyzed for hematocrit (HCT),and hemoglobin content measurements, as well as measurements of thevarious blood cells, such as WBC, RBC, and platelets. The results arepresented in Tables 16. ISIS oligonucleotides that caused changes in thelevels of any of the blood cell counts outside the expected range forantisense oligonucleotides were excluded in further studies.

TABLE 16 Complete blood count in CD1 mice HCT Hemoglobin Platelets RBCWBC (%) (g/dL) (10³/μL) (10⁶/μL) (10³/μL) PBS 45 13.9 844 8.4 6.1 ISIS549372 42 13 425 8.4 2.9 ISIS 585233 34 10.2 877 6.5 14.8 ISIS 585259 3610.9 1055 7 9.8 ISIS 585262 44 13.3 497 8.5 7.5 ISIS 585263 45 14 7068.8 7.3 ISIS 585264 43 13.1 659 8.3 3.6 ISIS 585265 42 12.7 732 8.2 7.3ISIS 585268 44 13.3 872 8.3 6.8 ISIS 585269 46 14.2 730 8.9 6RNA Analysis

RNA was isolated from the liver and muscle. Mouse Target X mRNAexpression was analyzed by RT-PCR. The data is presented in Table 17.The results indicate that antisense oligonucleotides targeting Target Xinhibited the expression of Target X mRNA in treated mice compared tothe control group.

TABLE 17 % inhibition of Target X mRNA levels compared to the PBScontrol Liver (%) Muscle (%) ISIS 549372 99 97 ISIS 585262 98 98 ISIS585263 92 97 ISIS 585264 76 97 ISIS 585265 99 98 ISIS 585268 98 97 ISIS585269 99 98

We claim:
 1. A compound comprising: a modified oligonucleotideconsisting of 10 to 20 linked nucleosides, wherein the modifiedoligonucleotide has a motif selected from: BBB-(D)₈-BBBAA;BBB-(D)₇-BBBAA; BBB-(D)₈-BBBAAA; BBB-(D)₇-BBBAAA; BABA-(D)₈-ABAB;BABA-(D)₇-ABAB; ABAB-(D)₈-ABAB; ABAB-(D)₇-ABAB; AABB-(D)₈-BBAA;AABB-(D)₇-BBAA; BBB-(D)₉-BBA; BBB-(D)₈-BBA; BBB-(D)₇-BBA;ABBB-(D)₈-BBBA; ABBB-(D)₇-BBBA; ABBB-(D)₈-BBBAA; ABBB-(D)₇-BBBAA;AABBB-(D)₈-BBB; AABBB-(D)₇-BBB; AABBB-(D)₈-BBBA; AABBB-(D)₇-BBBA;ABBAABB-(D)₈-BB; ABBAABB-(D)₇-BB; ABBABB-(D)₈-BBB; and ABBABB-(D)₇-BBB;wherein each B is a bicyclic nucleoside, each A is a non-bicyclicmodified nucleoside, and each D is a 2′-deoxynucleoside.
 2. The compoundof claim 1, wherein at least one B is a cEt nucleoside.
 3. The compoundof claim 2, wherein each B is a cEt nucleoside.
 4. The compound of claim1, wherein at least one B is a LNA nucleoside.
 5. The compound of claim4, wherein each B is a LNA nucleoside.
 6. The compound of claim 1,wherein each B is either a cEt nucleoside or a LNA nucleoside.
 7. Thecompound of claim 1, wherein each B comprises the same bicyclic sugarmoiety.
 8. The compound of claim 1, wherein at least one A is a 2′-MOEnucleoside.
 9. The compound of claim 1, wherein at least one A is a2′-OMe nucleoside.
 10. The compound of claim 1, wherein each A comprisesthe same non-bicyclic modified sugar moiety.
 11. The compound of claim 1comprising at least one modified internucleoside linkage.
 12. Thecompound of claim 11 comprising at least one phosphorothioateinternucleoside linkage.
 13. A method of modulating expression of atarget nucleic acid in a cell comprising contacting the cell with acompound according to claim
 1. 14. The method of claim 13, wherein thecell is in an animal.
 15. The method of claim 14, wherein the animal isa human.
 16. A pharmaceutical composition comprising the compoundaccording to claim 1 and a pharmaceutically acceptable diluent.
 17. Amethod of modulating expression of a target nucleic acid in an animalcomprising administering to the animal a pharmaceutical compositionaccording to claim 16.